The invention relates to a novel DNA sequence, which encodes a previously unidentified lignin biosynthetic pathway enzyme, sinapyl alcohol dehydrogenase (SAD), that regulates the biosynthesis of syringyl lignin in plants. Methods for incorporating this novel SAD gene sequence or sequences similar to this SAD gene into plant genome for genetic engineering of syringyl-enriched lignin in plants are also provided.
Lignin, a complex phenolic polymer, is a major part of the supportive structure of most woody plants including angiosperm and gymnosperm trees which, in turn, are the principal sources of fiber for making paper and cellulosic products. Lignin generally constitutes about 25% of the dry weight of the wood, making it the second most abundant organic compound on earth after cellulose. Lignin provides rigidity to wood for which it is well suited due, in part, to its resistance to biochemical degradation.
Despite its importance to plant growth and structure, lignin is nonetheless problematic to post-harvest, cellulose-based wood/crop processing for fiber, chemical, and energy production because it must be removed or degraded from cellulose at great expense. Certain structural constituents of lignin, such as the guaiacyl moiety, promote monomer cross-linkages that increase lignin resistance to degradation (Sarkanen, 1971; Chang and Sarkanen, 1973; Chiang and Funaoka, 1990). In angiosperms, lignin is composed of a mixture of guaiacyl and syringyl monolignols, and can be degraded at considerably less energy and chemical cost than gymnosperm lignin, which consists almost entirely of guaiacyl moieties (Freudenberg, 1965). It has been estimated that, if syringyl lignin through genetic engineering, could be incorporated into gymnosperm guaiacyl lignin or into angiosperms to increase the syringyl lignin content, the annual saving in processing of such genetically engineered plants as opposed to their wild types would be in the range of $6 to $10 billion in the U.S. alone. Consequently, there has been long-standing incentive to understand the biosynthesis of syringyl monolignol to genetically engineer plants to contain more syringyl lignin, thus, facilitating wood/crop processing (Trotter, 1990; Bugos et al., 1991; Boudet et al., 1995; Hu et al., 1999).
Although it has been known that syringyl lignin is derived from guaiacyl lignin, only a partial syringyl monolignol pathway and its genes encoding the enzymes that catalyze the steps of the pathway have been uncovered. This partial syringyl monolignol pathway, which diverges from guaiacyl pathway at coniferaldehyde, is mediated by genes encoding the enzymes coniferyl aldehyde 5-hydroxylase (CAld5H) (Osakabe et al., 1999) and S-adenosyl-L-methionine (SAM)-dependent 5-hydroxyconiferaldehyde O-methyltransferase (AldOMT) (Li et al., 2000), respectively, for the formation of sinapaldehyde (see, FIG. 1). However, sinapaldehyde must be enzymatically converted into sinapyl alcohol, the syringyl monolignol, for the biosynthesis of syringyl lignin in plants (see, FIG. 1). The gene encoding such an enzyme, i.e., a sinapyl alcohol dehydrogenase (SAD), has never been identified nor cloned and represents a missing gene that is indispensable for genetic engineering of syringyl lignin in plants. Thus, there is a need for the identification and isolation of this key gene (SAD) for genetic augmentation of syringyl monolignol biosynthesis in plants and for the methods for such genetic manipulation comprising the use of this key gene.